PROJECT SUMMARY The intricate and dynamic balance between self-renewal, proliferation and differentiation of stem/progenitor cells of the salivary gland (SG) must be tightly regulated to ensure proper morphogenesis, homeostasis and regeneration. Alterations to normal SG function, clinically often manifested with hyposalivation, are associated with diseases such as Ectodermal Dysplasias, Sjgren?s Syndrome and with ?- irradiation therapy of oral cancer patients. Currently, treatment options for hyposalivation remain limited. Therefore, identifying crucial transcriptional and signaling networks that govern stem/progenitor cell function of the SG are much needed to facilitate targeted stem cell and regenerative-based therapies. ?Np63, a stem cell enriched transcription factor, plays a critical role in epithelial regenerative function as evident from the phenotype of ?Np63-null animals which exhibit developmental arrest and agenesis of epithelial-rich organs including the SG. However, our current knowledge of the molecular mechanisms by which ?Np63 directs gene expression programs necessary for the commitment, maintenance and differentiation of the stem/progenitor cell population in the SG is lacking. Thus, identifying the p63-driven regulatory networks, particularly in the global and genomic context, is a key step towards a better understanding of the biology of SG stem/progenitor cells and ultimately in directing new strategies in treating SG dysfunction. To address these knowledge gaps, we will utilize multiple versatile mouse models to study two major independent areas of interest. First, we will use conditional knockout mouse models to examine the role of ?Np63 in SG morphogenesis and in adult tissue maintenance and repair (Aim1). Such systematic studies are much needed as they will identify for the first time, the functional role of p63 in SG development and in orchestrating stem/progenitor cell differentiation programs. Second, we will use p63 knockout mouse models and lineage tracing experiments to determine the contribution of p63+ stem and progenitor cells during SG regeneration and in response to irradiation induced damage. Furthermore, we will define p63 dependent SG cellular identities and the defined cellular and molecular signature that is associated with regeneration and in response to irradiation by performing single cell RNA-sequencing (Aim2). These studies will better elucidate the role of ?Np63 in SG organogenesis, and adult gland maintenance, and elucidate its contribution towards SG regeneration and in response to irradiation induced injury. Importantly, our genetic and genomic studies will also uncover novel ?Np63-pathways dependent and independent biomarkers and drivers of the distinct cell states associated with regeneration and radiosensitivity. Long term, knowledge garnered from our proposed mechanistic studies will have clinical and therapeutic implications for human patients who suffer from SG dysfunction diseases.